Cysteine imaging reveals early redox dysregulation and identifies gnetol as a ferroptosis-modulating agent in doxorubicin cardiotoxicity

Figures

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  Fig. 1. Design, synthesis, and photophysical characterization of the cysteine-responsive fluorescent probe CCP. (A) Chemical structures of CCP and its cysteine-activated product CCP-OH, illustrating the thiol–Michael addition and cleavage mechanism responsible for fluorescence activation. (B) Absorption and fluorescence emission spectra of CCP (10 μM) in the absence or presence of cysteine (100 μM). (C) Time-dependent fluorescence response of CCP (10 μM) upon addition of cysteine (100 μM). (D) Fluorescence emission spectra of CCP (10 μM) in the presence of increasing concentrations of cysteine (0–200 μM). (E) Linear relationship between fluorescence intensity at 615 nm and cysteine concentration. (F) Fluorescence response of CCP-OH under different pH conditions. (G) Selectivity of CCP toward cysteine over other biologically relevant analytes, including biothiols, amino acids, reactive oxygen species, and metal ions. All measurements were performed in PBS buffer (10 mM, pH 7.4, 1% DMSO) at 37 °C. Data are presented as mean ± SD (n = 3).

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  Fig. 2. Validation of CCP for intracellular cysteine imaging and early in vivo detection of doxorubicin-induced cardiotoxicity. (A) Cell viability of H9C2 cells after incubation with increasing concentrations of CCP, assessed by CCK-8 assay (n = 4). (B) Confocal fluorescence images of H9C2 cells stained with CCP under basal conditions, after thiol depletion with NEM, or after supplementation with exogenous Cys. (C) Quantification of intracellular CCP fluorescence intensity corresponding to (B) (n = 3). (D) Cell viability of H9C2 cells treated with increasing concentrations of DOX (n = 4). (E) Confocal fluorescence images of H9C2 cells stained with CCP after DOX treatment. (F) Quantitative analysis of CCP fluorescence intensity corresponding to (E) (n = 3). (G) In vivo fluorescence imaging of mice following tail-vein injection of CCP (1.5 mg/kg) at 30 min post-injection. (H) Quantification of cardiac fluorescence signals shown in (G) (n = 3). (I) Representative M-mode echocardiographic images of control and DOX-treated mice. (J) Quantitative analysis of left ventricular ejection fraction (EF) and fractional shortening (FS) (n = 3). Scale bars: 20 μm. Data are presented as mean ± SD. ∗P <…

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  Fig. 3. CCP-based screening identifies gnetol as an effective modulator of intracellular cysteine levels. (A) Schematic illustration of the CCP-guided screening strategy for identifying compounds that enhance intracellular cysteine under DOX stress. (B) Primary screening results of candidate compounds based on CCP fluorescence intensity (n = 3). (C) Cytotoxicity evaluation of the four candidate compounds in H9C2 cells. (D) Effects of candidate compounds on cell viability in DOX-treated H9C2 cells (n = 3). (E) Chemical structure of gnetol. (F) Confocal fluorescence images of CCP-stained H9C2 cells treated with gnetol under DOX challenge. (G) Quantitative analysis of intracellular CCP fluorescence intensity corresponding to (F) (n = 3). Scale bars: 100 μm. Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001,∗∗∗P < 0.0001.

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  Fig. 4. Gnetol suppresses DOX-induced ferroptotic injury in H9C2 cardiomyocytes. (A) Mitochondrial membrane potential assessed by JC-1 staining. (B) Quantification of JC-1 red/green fluorescence ratio. (C) Lipid peroxidation levels were quantified by flow cytometry following C11-BODIPY staining (n = 3). (D) Representative fluorescence images of cells stained with CM-H2DCFDA (green) to indicate cytosolic ROS and Hoechst 33342 (blue) to label nuclei. Scale bar, 50 μm. (E) Quantification of CM-H2DCFDA mean fluorescence intensity presented as fold change relative to CON (n = 3). (F) Intracellular Fe2+ levels detected using a Fe2+-sensitive fluorescent probe (n = 6). (G) Relative mRNA expression levels of ferroptosis- and cysteine-related genes (n = 3). (H) Western blot analysis of phosphorylated SMAD1/5/9, total SMAD1/5/9, ferroportin (FPN1), and GPX4. (I) Quantitative analysis of protein expression shown in H (n = 3). Data are presented as mean ± SD (n ≥ 3). ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001.

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  Fig. 5. Gnetol ameliorates doxorubicin-induced cardiomyopathy in vivo. (A) Experimental scheme of the DOX-induced cardiomyopathy model and gnetol treatment. (B) Changes in body weight during the treatment period. (C) Heart weight-to-tibia length ratio (HW/TL). (D) Representative gross morphology of hearts from each group. (E) Representative M-mode echocardiographic images. (F) Quantitative analysis of cardiac function parameters. (G) Representative histological images of heart sections stained with H&E, Masson's trichrome, and wheat germ agglutinin (WGA). (H) Quantification of myocardial fibrosis area. (I) Quantification of cardiomyocyte cross-sectional area. Data are presented as mean ± SD (n = 6-8). ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001.

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  Fig. 6. Gnetol attenuates myocardial ferroptotic injury in vivo by modulating the SMAD–hepcidin–FPN1 axis. (A) Relative mRNA expression levels of hepcidin in cardiac tissues. (B) Representative immunohistochemical staining of hepcidin in heart sections. (C) Quantitative analysis of hepcidin-positive staining area. (D) Western blot analysis of phosphorylated SMAD1/5/9, total SMAD1/5/9, hepcidin, FPN1, and GPX4 in cardiac tissues. (E) Densitometric quantification of protein expression shown in (D). (F) Transmission electron microscopy images showing mitochondrial ultrastructure. (G) Representative immunofluorescence images of 4-hydroxynonenal (4-HNE) staining. (H) Quantitative analysis of 4-HNE fluorescence intensity. (I–L) Serum biochemical indicators of oxidative injury and myocardial damage. (M, N) Levels of GSH and GSSG in cardiac tissues. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001.